PROJECT SUMMARY/ABSTRACT In the United States (US), diagnosis of spotted fever rickettsiosis leads to a prognosis ranging from good to guarded, depending on the causative agent and associated disease. Of the tick-borne spotted fever group Rickettsia (SFGR) that cause disease in the US, Rickettsia rickettsii, the agent of Rocky Mountain spotted fever (RMSF), is the most pathogenic. A second pathogenic SFGR in the US, Rickettsia parkeri, was confirmed over nearly 15 years ago. Rickettsia parkeri rickettsiosis, which overlaps in range with RMSF, results in milder disease. Rickettsia 364D, in the western US, is now the third tick-borne SFGR associated with human disease. The remaining SFGR in hard ticks (Ixodidae) in the US are considered non-pathogenic or of unknown or potential pathogenicity. The extent to which exposure to SFGR that are not considered pathogenic stimulates an immune response that affects infection or clinical disease from pathogenic SFGR is unclear. Our understanding of host immunity would benefit from studies using an tick-SFGR-vertebrate host transmission model that exploits a tick vector known to harbor both a pathogenic and non-pathogenic SFGR and animal model known to demonstrate clinical rickettsiosis. Here, we use Amblyomma maculatum (Gulf Coast tick), and its associated rickettsiae, R. parkeri, a pathogenic SFGR that can be studied under biosafety level two, ?Candidatus Rickettsia andeanae?, a SFGR that is transmissible but considered non-pathogenic, and a guinea pig model. Additionally in this project, we address challenges in distinguishing antibodies to the pathogenic SFGR that overlap in geographical range. Our long-term goal is to evaluate the role of vertebrate host immunity in response to SFGR infection. Our overall objective in this COBRE Phase II project is to clarify primary immune system contributions after SFGR exposure via tick transmission. Specifically, we will determine how prior SFGR exposure influences infection and clinical disease from the pathogenic SFGR using a guinea pig model. Additionally, we will test the utility of Rickettsia species-specific antigens in a diagnostic assay. Our central hypothesis is that successful establishment of R. parkeri infection is determined by both the lack of an effective innate immune response and the lack of an adaptive response to prior SFGR exposure. Our rationale is that innate immunity, the first line of defense against tick transmitted disease, is not sufficient to prevent R. parkeri infection of endothelial cells proximal to the tick bite. We have detected an antibody response to SFGR in animals exposed to ?Candidatus Rickettsia andeanae,? the non-pathogenic SFGR also found in A. maculatum, and believe this may prevent establishment of and disease by the pathogenic SFGR, R. parkeri, in vertebrates. Our proposed research is significant because it will provide insight into the contributions of prior SFGR exposure to development of disease, and thus risk of spotted fever rickettsiosis. We believe this is innovative by combining existing expertise in the A. maculatum-SFGR system and recent additional expertise in immunology to create a synergy that addresses immune protection and the need to specifically distinguish causative agents of spotted fever rickettsiosis using serologic assays.